The present disclosure concerns a method and system for imaging a molecular strand, e.g. a molecular complex such as DNA.
The interaction between nucleic acids (e.g. DNA and RNA) and proteins plays an important role in the molecular biology of the cell, being at the heart of DNA replication, transcription, organization, and repair. Not only is understanding these processes important to understanding life in itself; it is also desired for generating key insights into disease mechanisms. Fluorescence microscopy and force spectroscopy using optical tweezers are two pillars of single-molecule research.
Optical tweezers are used for measurement of global mechanical and structural properties of DNA-protein complexes. These measurements can be related to coinciding fluorescent emissions. For example, Lang et al. (Nat. Methods 1, 2004, 133-139) describe simultaneous, coincident optical trapping and single-molecule fluorescence wherein mechanical transitions in the structure of DNA are probed by analyzing fluctuations in fluorescence intensity within a fixed confocal volume.
Fluorescence microscopy can provide local structural information. For example, Candelli et al. (Phys. Chem. Chem. Phys., 2011, 13, 7263-7272) describe a method integrating optical trapping with micro-fluidics and single-molecule fluorescence microscopy to study heterogeneous/complex protein interactions. This combination of wide-field fluorescence microscopy and optical tweezers allows localization of labelled proteins on optically stretched DNA with sub-10 nm (nanometer) precision at tensions above 1 pN (pico-Newton).
US 2011/0201509 by Tegenfeldt discloses a method for the mapping of the local AT/GC ratio along the DNA in which the DNA is denaturated to partially melt a double-stranded DNA molecule depending e.g. on temperature. The method includes staining DNA with a fluorescent dye, the emission of which is sensitive to whether the DNA is single stranded or double stranded. The DNA is introduced with a flow into a nanochannel device that if necessary stretches the DNA to prevent overlap of different segments of the molecule. The basic tool for observing the resulting pattern of the DNA is diffraction limited standard fluorescence microscopy. Once raw movies of denatured molecules are acquired, a software program is used to align the barcode fluorescence pattern across the images as a function of time.
US 2012/0002031 by Pertsinidis discloses a microscope that provides sub-nanometer resolution in measurements of molecular-scale distances using far-field fluorescence imaging optics. This performance is achieved using feedback control of the position of individual fluorescent molecules, allowing collection of >106 photons locked at the same position. The imaging system is calibrated by raster scanning a fluorescent emitter and correcting imperfections at pixel and subpixel scales by comparing the position of the fluorescence image and the known displacement of the sample, using a sub-nm accurate piezo translation stage. A sample arrangement is illustrated in which an end of a DNA molecule is attached to a surface and the other end is held in an optical trap.
There is yet a desire for a method and system providing improved control and accuracy for the imaging of a molecular strand.